The latent reservoir for HIV-1 in resting CD4+ T cells is a major barrier to virus eradication. There is great current interest in potential strategies for eliminating this reservoir. During the previous funding period, we have carried out mechanistic studies that have led to a reevaluation of current models of latency and have shown that mechanisms of latency are most accurately analyzed in primary cell systems rather than in transformed T cell lines. We developed an in vitro system in which primary resting CD4+ T cells carrying latent HIV-1 genomes can be generated. In this proposal, we describe the use of this system to identify and characterize small molecules capable of reactivating latent HIV-1 for use in efforts to eradicate this reservoir. We have already identified one promising hit which we will characterize mechanistically. In addition, we will use this system to explore key unanswered questions regarding the dynamics of the latent reservoir, focusing on issues that are important for eradication strategies. The Specific Aims are (1) to determine the mechanism by which the identified hit (5-hydroxy-1,4-naphthoquinone) reactivates latent HIV-1, (2) to use the novel latency model in primary resting CD4+ T cells to screen selected small molecule libraries for other agents that reactivate latent HIV-1 without inducing global T cell activation, (3) to carry out a comparative in vitro evaluation of the efficacy of agents that reactivate latent HIV-1, and (4) to use the primary cell model to explore mechanisms involved in the maintenance of HIV-1 latency.